A re‐examination of the circumscription of Saxifraga mengtzeana (Saxifragaceae)

Abstract In the Flora of China account of Saxifraga mengtzeana Engl. & Irmsch., eight synonyms were attributed to it and one variant, recognized as Saxifraga epiphylla Gornall & Ohba, was split from it. This study reevaluates the taxonomic status of some of the synonyms and of the segregated species in light of new evidence presented here. Morphological and molecular evidence demonstrate that collections from northwestern Yunnan and Sichuan are genetically differentiated from those in southeastern Yunnan and neighboring Guangxi. Observations in the field and in cultivation show that the peltate petiole attachment diagnostic of S. mengtzeana var. peltifolia Engl. & Irmsch. is developmentally labile. Similar observations combined with molecular data show that viviparous phenotypes, formerly treated as S. epiphylla, although largely under genetic control, occur sporadically throughout the ranges of both northern and southern taxa. Collections from northwestern Yunnan and Sichuan are best recognized as Saxifraga geifolia Balf.f., whereas those from southeastern Yunnan and neighboring Guangxi are S. mengtzeana. Peltate‐leaved variants of the latter are given no status and are relegated to complete synonymy. Viviparous phenotypes of S. mengtzeana and S. geifolia are recognized at the rank of forma.


| INTRODUC TI ON
Saxifraga L., the largest genus in Saxifragaceae, comprises more than 440 species that are widely distributed throughout arctic and montane regions of the Northern Hemisphere (Ebersbach et al., 2017;Pan et al., 2001;Tkach et al., 2015). Recent molecular phylogenetic research recognized at least 13 sections and nine subsections within the genus (Tkach et al., 2015). Saxifraga sect. Irregulares Haw., characterized by asymmetric flowers with two unequally elongated and three short petals, was one of the earliest lineage of Saxifraga to diverge (Magota et al., 2021;Soltis et al., 2001;Tkach et al., 2015;Zhang et al., 2020). It is distributed in eastern Asia, and many species are narrow endemics only known from a few localities. (Magota et al., 2021;Pan et al., 2001). Section Irregulares currently comprises 20 species, including seven recently described from China (14 species in China currently). (Wang et al., 2008;Zhang et al., 2017Zhang et al., , 2018Zhang et al., , 2019Zhang et al., , 2021Zhang et al., , 2022Zhao et al., 2019). The diversity of S. sect.
Irregulares was previously underestimated as only seven species of S. sect. Irregulares were recorded in Flora of China (Pan et al., 2001), and more investigations and phylogenetic analyses are needed to clarify the patterns of variation and the delimitation of species (Zhang et al., 2021). The purpose of this study was to reevaluate the taxonomic and phylogenetic status of taxa that were associated with or synonymized under S. mengtzeana Engl. & Irmsch. in the account of this species in Flora Reipublicae Popularis Sinicae (Pan, 1992) and in Flora of China (Pan et al., 2001). Saxifraga henryi Balf.f. (1916) were described based on the same collection (A. Henry 9118), only S. mengtzeana needs to be considered of these on species level (Balfour, 1916;Engler & Irmscher, 1913;Gornall et al., 2000;Pan et al., 2001; Taxonomic treatment section).
Variants with a foliar embryo in the basal leaf sinus were not mentioned by Engler & Irmscher (1913, 1916) nor by Balfour (1916), but they seem to have been first recognized by Pan (1992), who misapplied the name S. aculeata to them. The type collections of S. aculeata, however, lack foliar embryos and, as explained earlier, are the same taxon as the type of S. mengtzeana (Gornall et al., 2000).
The viviparous taxon was distinguished as a separate species and given its first formal name as S. epiphylla Gornall & H. Ohba (Gornall et al., 2000) based on the collection by Feng 12638 (Figure 1b). Soon afterwards, Chuang (2001) (Pan et al., 2001), although morphologically, the leaf shape of the holotype specimen is rounder than the triangular-cordate to ovate leaf shape of S. mengtzeana. Geographically, the type locality of S. geifolia is some 500 km distant from the type locality of S. mengtzeana. The morphological and geographical differences suggest that S. geifolia may be distinct rather than conspecific with S. mengtzeana.

| Phylogenetic reconstruction
We sampled 22 collections representing Saxifraga mengtzeana and related taxa, of which 20 were newly sequenced and two were obtained from GenBank (Table 1). Leaf materials were collected in the field and from dried herbarium specimens, voucher information and GenBank accession numbers were presented in Table 1. Saxifraga sinomontana from Saxifraga sect. Ciliatae was selected as the outgroup, based on previously proposed phylogenetic relationships based on molecular analyses (Tkach et al., 2015).
Total genomic DNA was extracted from leaf material using DP305 Plant Genomic DNA kits (Tiangen, Beijing, China) following the manufacturer's protocol. Sequencing libraries were generated using the NEB Next® Ultra DNA Library Prep Kit for Illumina® (NEB, USA). The prepared libraries were sequenced on an Illumina Hiseq 4000 platform with 150 bp paired-end reads. Plastid genome data and nrDNA sequences were assembled using GetOrganelle pipeline (Jin et al., 2020). Complete plastomes were annotated in batches using PGA v.3 (Qu et al., 2019). A concatenation-based approach was conducted for the plastid coding sequences for 73 shared protein coding genes of 22 samples, and sequences were aligned in MAFFT 7 (Katoh et al., 2019). Partial nrDNA sequences include internal transcribed spacers ITS1 and ITS2 of nuclear ribosomal DNA and the 5.8 S rRNA gene (ITS region). Phylogenetic reconstruction was performed using maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood analysis was implemented in IQ-Tree with 1000 bootstrap (BS) replicates to assess clade support (Nguyen et al., 2015). As identified by jModeltest 2.1.7, the GTR + I + G and SYM + G model of sequence evolution was selected using the Akaike information criterion (AIC) for BI analyses of plastid dataset and ITS sequences, respectively (Posada, 2008).
Bayesian analysis used MrBayes version 3.2.6 (Huelsenbeck & Ronquist, 2001). Four parallel Markov Chains Monte Carlo (MCMC) simulations were run and sampled every 1000 generations for 20 million generations in total, with the first 25% trees discarded as burn-in, and runs were considered to have converged to stationarity when their average standard deviation of split frequencies was <0.01. (Ronquist et al., 2012).

| Morphological comparison
Morphological data were recorded from field collections, cultivations, and herbarium specimens. Voucher specimens of our collections were deposited in the herbarium of the Kunming Institute of Botany org/). Leaf length relative to width (aspect ratio) and the angle of leaf apex ( Figure 3) were measured for quantitative analysis (Appendix S1).
In addition, observations were made of the base of leaf blade (cordate or peltate, viviparous or not), the shape of the leaf apex (subacute vs. obtuse), the leaf texture (thick, leathery and slightly succulent vs. papery or slightly leathery), and markings on the adaxial leaf surface (concolorous vs. with white or virescent streaks). Voucher specimens for the morphological observations are cited under Additional specimens examined in the taxonomic treatment and in Table 1. Besides, in the ITS phylogeny, Saxifraga viridiflora was sister to the clade of all species mentioned above, rather than sister to S. geifolia in plastid phylogeny ( Figure S1).

| Morphology
Analysis of the morphological variation shows that it can be divided into two reasonably well-marked groups (Figure 3) based on leaf outline: (a) a northern group with rotund to ovate, more or less isodiametric leaves with bluntly crenate margins, and (b) a southeastern group with triangular-ovate leaves, longer than wide, with more coarsely dentate margins. Plants with a peltate petiole insertion are restricted to the southeastern group, but those with foliar embryos occur in both groups (Figure 3).
In addition to the differences in leaf shape, other distinctive features associated with the geographical groups include the leaf apex (subacute in the south vs. obtuse in the north), leaf texture (thick, leathery, and slightly succulent in the south vs. papery or slightly leathery in the north), and markings on the adaxial leaf surface (concolorous in the south vs. with white or virescent streaks in the north) Appendix S1). Field studies at the type locality of S. epiphylla in Malipo County, Yunnan, showed that, even here, the population is polymorphic for this feature. Sometimes, the embryos are very small, reduced to a tiny, leafless bud. Furthermore, we found foliar embryos in some plants in PingBian County, which is adjacent to Mengtze, the type locality of S. mengtzeana (Figure 7). Examination of specimens of these two species, including the types, shows very little difference apart from the foliar embryos ( Figure 1, Table 2).

| DISCUSS ION
Morphological and molecular patterns of variation are consistent in distinguishing a northern taxon from a southeastern taxon. The northern taxon corresponds to Saxifraga geifolia, with rotund to ovate, more or less isodiametric, cordate leaves with bluntly crenate margins, an obtuse leaf apex, a papery or only slightly leathery texture, and whose adaxial surface is streaked with white or pale green.
The southeastern taxon matches S. mengtzeana, with triangularovate (longer than wide), cordate leaves, with more coarsely dentate margins and a subacute apex, a thick, leathery and slightly succulent texture, and a concolorous adaxial surface. Despite some morphological overlap between the northern and southeastern populations, the differences are sufficient to warrant the recognition of two distinct species, particularly in light of molecular support. Although significant cytonuclear discordances were inferred,  found no features to support their separation except the presence of foliar embryos. Molecular data also strongly suggest that plants with foliar embryos do not form a monophyletic group (Figure 4). Since the character appears to be under genetic control and is an important feature of the phenotype, scattered within populations, we propose that taxonomic recognition is warranted at the rank of forma.
This is easily accomplished in the case of S. mengtzeana where a valid description and a type specimen exists, but a new taxon needs to be established for the viviparous plants of S. geifolia.